Synapses of the mammalian CNS are highly specialized cellular junctions designed for rapid and regulated signaling between nerve cells and their targets. Morphologically, synapses are asymmetric structures composed of a presynaptic bouton filled with synaptic vesicles (SVs), a synaptic cleft and a postsynaptic specialization. Proper synaptic function requires the precise functional integration of numerous, appropriate localized components. However, little is known about the molecular and cellular mechanisms underlying the assembly of CNS synapses. Recent advances in determining the molecular composition of CNS synapses has allowed neuroscientists to address a number of fundamental questions regarding the assembly and function of CNS synapses. In our recent studies, we have begun to analyze how presynaptic active zones (AZs) are formed. These studies have lead to the discovery of two AZ cytoskeletal proteins, Piccolo and Bassoon, which appear to be central organizers of these sites of synaptic vesicle exocytosis and neurotransmitter release. More recently we have examined when, how and in what form these AZ proteins are transported and recruited into nascent synapses. These studies have answered several basic neurobiological questions regarding the timing of synaptogenesis and whether AZ formation precedes the formation of the postsynapse. In particular, we have found that glutamatergic synapses form in less than two hours and that AZs form prior to the postsynapse. Mechanistically, we could show that the rapid formation of AZs (less than 20 mins) appears to be accomplished by the fusion of a small number of AZ precursor vesicles carrying numerous components of the AZ that are essential for SV exocytosis. Experiments outlined in this proposal will test the hypothesis that these putative AZ precursor vesicles are not only essential for the assembly of functional AZs, but also for the formation of a functional postysynapse. This will be accomplished by determining the molecular composition of these precursor vesicles, whether their fusion at nascent synaptic sites is required for AZ formation and whether they facilitate the release of factors that promote the clustering of postsynaptic glutamate receptors and structural proteins. These studies are critical to the understanding of how synapses are formed during normal development, as well as in developmental disorders and degenerative diseases.